Mitochondria are dynamic and complex organelles that play a central role in all aspects of biology, including energy production, intermediary metabolism, and apoptosis. These broad cellular functions also place mitochondria as a central player in human health. Mitochondrial dysfunction is associated with a wide range of diseases, including cancer, type 2 diabetes, and most neurodegenerative disorders. As a result of these wide-ranging critical activities, many efforts have focused on identifying and characterizing the mitochondrial proteome, with over 1,000 proteins identified to date in mammals. Remarkably, however, roughly one-quarter of these proteins remain essentially uncharacterized. These include many proteins that are highly conserved throughout eukarya, a strong indication that they perform a fundamentally important function. Our prior studies of two uncharacterized mitochondrial conserved proteins (MCPs) support this proposal, revealing new roles for these proteins in critical aspects of mitochondrial function and, in one case, providing a direct link to an inherited form of cancer. We propose to continue these functional studies of the mitochondrial proteome using genetic and biochemical approaches in yeast, Drosophila, mammalian cells, and mice. The three specific aims in this proposal each focus on a different critical aspect of mitochondrial function: (1) To determine the role of the BRP44 protein family in carbohydrate metabolism, (2) To determine the role of C6orf57 protein family in maintaining energy homeostasis, and (3) To determine the role of HIG proteins in mitochondrial III2IV2 respiratory super complex assembly. We will undertake genetic and biochemical studies in yeast focused on defining the mechanistic function of each MCP. These studies will be extended through genetic and metabolic studies in Drosophila, in close coordination with our yeast work, to determine the biological and physiological activity of each MCP in the context of an intact developing animal. Functional studies in mammalian cells will be used to test the hypothesis that specific MCP activities have been conserved through evolution. Finally, selected mouse models will be generated to enable elucidation of protein function in mammalian physiology. Our goal in this research will be to provide a new understanding of the biochemical and cellular function of each MCP, determine how they contribute to normal mitochondrial activity, and, whenever possible, link MCP function to human health and disease.